The bivalent genetic engineering vaccine of Japanese encephalitis (JE) and Aujeszkj disease (AD) was developed to provide a novel approach to prevent and control these two diseases. NS1 gene of Japanese encephalitis virus (JEV) SA14-14-2 strain was produced by reverse transcriptase-mediated PCR (RT-PCR) and was cloned into vector pUSK to form recombinant plasmid (designed as pUSK-NS1). A co-transfection experiment was performed in porcine kidney (PK-15) cells with pUSK-NS1 and the genome of the vector virus (PRV TK(-)/gG(-)/LacZ(+) mutant). By plaque purification, PCR detection and southern hybridization, recombinant pseudorabies virus (PRV) expressing NS1 protein of JEV was acquired and named TK(-)/gG(-)/NS1(+). Western blot analysis and ELISA demonstrated the NS1 protein expression. To evaluate the recombinant virus's potential application, we characterized the safety and immune responses in Balb/c mice and swine. The safety test indicated that, when receiving the recombinant virus at a concentration of 10(6.0)pfu, no virulence of the recombinant virus to the mice, piglets and pregnant sows was observed. The vaccinated animals could acquire protective immunity against lethal challenge of the virulent PRV Ea strain and develop a good humoral and cellular immune response against JEV. The above results revealed that the recombinant virus could be a suitable candidate vaccine strain for developing a novel genetic vaccine to combat pseudorabies and Japanese encephalitis in the pig industry.